Phosphotriesterase from agrobacterium radiobacter p230

ABSTRACT

The present invention provides enzymes capable of hydrolysing organophosphate (OP) molecules. In particular, the invention provides a phosphotriesterase enzyme identified from an  Agrobacterium radiobacter  strain isolated from soil that hydrolyses OP pesticides, and the gene encoding that enzyme. The invention also provides mutants of the identified phosphotriesterase enzyme which have altered substrate specificity. The use of these enzymes in bioremediation strategies is also provided.

FIELD OF THE INVENTION

This invention relates to enzymes capable of hydrolysing organophosphate(OP) molecules. In particular, the invention relates to aphosphotriesterase enzyme identified from an Agrobacterium radiobacterstrain isolated from soil that hydrolyses OP pesticides, and the geneencoding that enzyme. The invention also relates to mutants of theidentified phosphotriesterase enzyme which have altered substratespecificity.

BACKGROUND OF THE INVENTION

Residues of organophosphate insecticides are undesirable contaminants ofthe environment and a range of commodities. Areas of particularsensitivity include contamination of soil, irrigation tailwater that isre-cycled, used by irrigators downstream or simply allowed to runoff-farm, and residues above permissible levels in agricultural andhorticultural exports. Poisoning with organophosphates presents aproblem for agricultural workers that are exposed to these chemicals, aswell as military personnel exposed to organophosphates used in chemicalwarfare. Furthermore, the stockpiling of organophosphorus nerve agentshas resulted in the need to detoxify these stocks. Bioremediationstrategies are therefore required for eliminating or reducing theseorganophosphate residues and/or stockpiles.

One proposed strategy involves the use of enzymes capable ofimmobilising or degrading the organophosphate residues. Such enzymes maybe employed, for example, in bioreactors through which contaminatedwater could be passed, or in washing solutions after post-harvestdisinfestation of fruit, vegetables or animal products to reduce residuelevels and withholding times. Suitable enzymes for degradingorganophosphate residues include OP hydrolases from bacteria (Mulbry,1992; Mulbry and Kearney, 1991; Cheng et al., 1999; U.S. Pat. No.5,484,728; U.S. Pat. No. 5,589,386), vertebrates (Wang et al., 1993;1998; Gan at al, 1991; Broomfield et al., 1999) and OP resistant insects(WO 95/19440 and WO 97/19176). It is desirable that the OP hydrolasesdegrade the organophosphate residues at a rapid rate.

The most thoroughly studied OP degrading enzyme is bacterialorganophosphate dihydrolase (OPD), which is encoded by identical geneson dissimilar plasmids in both Flavobacterium sp. ATCC 27551 andBrevundimonas diminuta MG (Harper of al., 1988; Mulbry and Karns, 1989).OPD is a homodimeric protein that is capable of hydrolysing a wide rangeof phosphate triesters (both oxon and thion OPs) (Dumas et al., 1989a,b). Its reaction mechanism directly or indirectly involves metal ions,preferably Zn⁺⁺. OPD has no detectable activity with phosphatemonoesters or diesters (Dumas et al., 1989a, b; 1990).

OPD homologues (phosphotriesterase homology proteins, or PHPs) have beenidentified in the genomes of Escherichia coli (ePHP), Mycobacteriumtuberculosis (mtPHP) and Mycoplasma pneumoniae (mpPHP), although onlyePHP has been tested for phosphotriesterase activity (Scanlan and Reid,1995; Buchbinder et al., 1998). No activity was detected in ePHP crudelysates with any of the substrates tested, such as p-nitrophenylacetate, bis(p-nitrophenyl) phosphate, paraoxon and p-nitrophenylphosphate.

OPD homologues have also been identified in vertebrates (Davies et al.,1997), although their function in these organisms is unknown. OPD, ePHP,mtPHP and mammalian PHPs are 27-30% identical at the amino acid level,while mpPHP is less similar. Amino acid residues involved in Zn⁺⁺binding are conserved across the six members of the phosphotriesterasefamily identified to date (Buchbinder et al., 1998).

Three other distinct OP hydrolysing enzymes have been isolated frombacteria with a history of exposure to OPs (Mulbry and Karns, 1989;Mulbry, 1992; Cheng et al., 1999). The two for which sequence data areavailable are unrelated to each other and to OPD. One, a prolidase fromAlteromonas sp., normally functions in hydrolysis of X-Pro dipeptides.Its activity for insecticidal OPs is reported as modest, although it hasnot been reported in terms of k_(cat)/K_(m) specificity constants (Chenget al., 1999). The other, an aryldialkylphosphatase (ADPase) fromNocardia sp. strain B-1, has a turnover number for ethyl parathion thatis 4500-fold lower than that reported for OPD (Mulbry and Karns, 1989;Mulbry, 1992).

Paraoxonase, or PON1, is a distinct OP hydrolysing enzyme found inmammals. Like OPD it is a metalloenzyme, preferring Ca⁺⁺ in this case,which is associated with low density lipoproteins in plasma and normallyinvolved in metabolism of oxidised lipid compounds (Gan et al., 1991;Sorenson et al., 1995). It has high activity for paraoxon, with aspecificity constant of around 10⁶ M⁻¹see (Doom et al., 1999; Hong andRaushel, 1999).

There is also evidence for other, so-called diisopropylfluorophosphatase (DFPase) enzymes in a wide range of vertebrates,invertebrates and microorganisms (Wang et al., 1998; Hoskin et al.,1999; Billecke et al., 1999). These enzymes are notably diverse in manyof their biochemical properties but are all characterised by theirhydrolytic activity against OP chemical warfare agents. Limited sequencedata suggest that they are unrelated to all the other OP hydrolyticenzymes described above.

OP resistant blowflies and houseflies have been the source of esteraseenzymes with activity against oxon OPs like chlorfenvinfos (CVP) andcarboxylester OPs like malathion (Newcomb et al., 1997; Campbell et al.1998; Claudianos et al. 1999; WO 95/19440; WO 97/19176). A Gly to Aspsubstitution at residue 137 in blowfly esterase E3 (and its houseflyortholog, ALI) resulted in the acquisition of activity for CVP, while aTrp to Leu/Ser mutation at residue 251 in the same enzyme resulted inactivity against malathion. However, the specificity constants of theseenzymes for their OP substrates are orders of magnitude less than thoseof OPD for paraoxon.

There is a need for further OP degrading enzymes which can be used inbioremediation strategies.

SUMMARY OF THE INVENTION

The present inventors have developed a rapid and sensitive fluorimetricassay for coumaphos (a thion OP insecticide) hydrolysis and used it toisolate a bacterium from contaminated soil that is capable of using OPsas the sole phosphorus source. 16S rDNA sequencing identified thebacterium (isolate P230) as a strain of Agrobacterium radiobacter. Thepresent inventors have also isolated and characterized the enzymeresponsible for this coumaphos hydrolytic activity and provide methodsfor the use of this enzyme in bioremediation strategies.

In one aspect, the present invention provides a substantially purifiedpolypeptide, the polypeptide being selected from:

-   -   (i) a polypeptide comprising a sequence provided in SEQ ID NO:1;    -   (ii) a polypeptide comprising a sequence provided in SEQ ID        NO:2;    -   (iii) a polypeptide comprising a sequence provided in SEQ ID        NO:3;    -   (iv) a polypeptide comprising a sequence provided in SEQ ID        NO:4; or    -   (v) a polypeptide comprising a sequence which is greater than        90% identical to any one of (i) to (iv),        wherein the polypeptide is capable of hydrolysing an        organophosphate molecule.

Preferred organophosphate molecules include, but are not limited to,coumaphos, coroxon, paraoxon, parathion, parathion-methyl, phosmet,fenthion, diazinon, chlorpyrifos, dMUP, DFP, dimethoate, malathion, andmalaoxon. More preferably, the organophosphate is phosmet or fenthion.

In a preferred embodiment, the polypeptide can be purified from anAgrobacterium sp.

In a further preferred embodiment, the polypeptide is at least 95%identical to any one of (i) to (iv), more preferably at least 97%identical, and even more preferably at least 99% identical to any one of(i) to (iv).

In another aspect, the present invention provides a substantiallypurified polypeptide, the polypeptide being selected from:

-   -   (i) a polypeptide comprising the sequence provided in SEQ ID        NO:1;    -   (ii) a polypeptide comprising the sequence provided in SEQ ID        NO:2; or    -   (iii) a polypeptide which is greater than 90% identical to (i)        or (ii).

In another aspect, a fusion polypeptide is provided which comprises apolypeptide according to the present invention fused to at least oneother polypeptide sequence.

Preferably, the at least one other polypeptide is selected from thegroup consisting of: a polypeptide that enhances the stability of thepolypeptide of the invention, and a polypeptide that assists in thepurification of the fusion polypeptide.

Preferably, the at least one other polypeptide is the maltose-bindingprotein.

In another aspect, the present invention provides an isolatedpolynucleotide, the polynucleotide comprising a sequence selected from:

-   -   (i) a sequence of nucleotides shown in SEQ ID NO:5;    -   (ii) a sequence of nucleotides shown in SEQ ID NO:6;    -   (iii) a sequence of nucleotides shown in SEQ ID NO:7;    -   (iv) a sequence of nucleotides shown in SEQ ID NO:8;    -   (v) a sequence encoding a polypeptide according to the present        invention; or    -   (vi) a sequence which is at least 90% identical to any one        of (i) to (v),        wherein the polynucleotide encodes a polypeptide capable of        hydrolysing an organophosphate molecule.

Preferably, the polynucleotide is at least 95% identical, morepreferably at least 97% identical, and even more preferably at least 99%identical to any one of (i) to (v).

In a further aspect, a vector is provided which comprises apolynucleotide according to the invention.

Preferably, the vector is suitable for the replication and/or expressionof a polynucleotide. The vectors may be, for example, a plasmid, virusor phage vector provided with an origin of replication, and preferably apromotor for the expression of the polynucleotide and optionally aregulator of the promotor. The vector may contain one or more selectablemarkers, for example an ampicillin resistance gene in the case of abacterial plasmid or a neomycin resistance gene for a mammalianexpression vector. The vector may be used in vitro, for example for theproduction of RNA or used to transfect or transform a host cell.

In another aspect, a host cell is provided which comprises a vectoraccording to the invention.

In a further aspect, the present invention provides a process forpreparing a polypeptide of the invention, the process comprisingcultivating a host cell of the invention under conditions which allowproduction of the polypeptide, and recovering the polypeptide. Suchcells can be used for the production of commercially useful quantitiesof the encoded polypeptide.

In another aspect, the present invention provides a composition forhydrolysing an organophosphate molecule, the composition comprising apolypeptide according to the invention, and one or more acceptablecarriers.

In another aspect, the present invention provides a composition forhydrolysing an organophosphate molecule, the composition comprising ahost cell of the invention, and one or more acceptable carriers.

It will be appreciated that the present invention can be used tohydrolyse organophosphates in a sample. For instance, after a crop hasbeen sprayed with an organophosphate pesticide, the organophosphateresidue can be hydrolysed from seeds, fruits and vegetables before humanconsumption. Similarly, organophosphate contaminated soil or water canbe treated with a polypeptide of the invention.

Accordingly, in a further aspect the present invention provides a methodfor hydrolysing an organophosphate molecule in a sample, the methodcomprising exposing the sample to a polypeptide according to theinvention.

Preferably, the polypeptide is provided as a composition of theinvention.

Further, it is preferred that the method further comprises exposing thesample to a divalent cation. Preferably, the divalent cation is zinc.

Preferably, the sample is selected from the group consisting of; soil,water, biological material, or a combination thereof. Preferredbiological samples include matter derived from plants such as seeds,vegetables or fruits, as well as matter derived from animals such asmeat.

Preferred organophosphate molecules include, but are not limited to,coumaphos, coroxon, paraoxon, parathion, parathion-methyl, phosmet,fenthion, diazinon, chlorpyrifos, dMUP, DFP, dimethoate, malathion, andmalaoxon. More preferably, the organophosphate is phosmet or fenthion.

The sample can be exposed to the polypeptide via any available avenue.This includes providing the polypeptide directly to the sample, with orwithout carriers or excipients etc. The polypeptide can also be providedin the form of a host cell, typically a microorganism such as abacterium or a fungus, which expresses a polynucleotide encoding thepolypeptide of the invention. Usually, the polypeptide will be providedas a composition of the invention.

Organophosphate molecules in a sample can also be hydrolysed by exposingthe sample to a transgenic plant which produces a polypeptide of thepresent invention.

Thus, in a further aspect a transgenic plant is provided which producesa polypeptide according to the invention.

In a further aspect, the present invention provides a method forhydrolysing an organophosphate molecule in a sample, the methodcomprising exposing the sample to a transgenic plant according to theinvention.

Preferably, the sample is soil.

Further, it is preferred that the polypeptide is at least produced inthe roots of the transgenic plant.

In yet another aspect, the present invention provides an isolated strainof Agrobacterium radiobacter deposited under NM01/21112 on 20 Apr. 2001at Australian Government Analytical Laboratories.

In another aspect, the present invention provides a composition forhydrolysing an organophosphate molecule, the composition comprising theAgrobacterium radiobacter strain of the invention, and one or moreacceptable carriers.

In yet another aspect, the present invention provides a method forhydrolysing an organophosphate molecule in a sample, the methodcomprising exposing the sample to an Agrobacterium radiobacter strainaccording to the invention.

The disclosure of the present invention can readily be used to isolateother bacterial species/strains which hydrolyse organophosphates. Forexample, other bacterial species/strains may be isolated using afluormetric screening method as disclosed herein. Alternatively, probesand/or primers can be designed based on the polynucleotides of thepresent invention to identify bacteria which produce naturally occurringvariants of the polypeptides of the present invention.

Accordingly, in a further aspect the present invention provides anisolated bacterium which produces a polypeptide according to theinvention.

Preferably, the bacterium is an Agrobacterium sp. More preferably, thebacterium is a strain of Agrobacterium radiobacter.

In a further aspect, the present invention provides the use of anisolated naturally occurring bacterium which produces a polypeptideaccording to the invention for hydrolysing an organophosphate in asample.

In a further aspect, the present invention provides a polymeric spongeor foam for hydrolysing an organophosphate molecule, the foam or spongecomprising a polypeptide according to the invention immobilized on apolymeric porous support.

Preferably, the porous support comprises polyurethane.

In a preferred embodiment, the sponge or foam further comprises carbonembedded or integrated on or in the porous support.

In a further aspect, the present invention provides a method forhydrolysing an organophosphate molecule in a sample, the methodcomprising exposing the sample to a sponge or foam according to theinvention.

In another aspect, the present invention provides a biosensor fordetecting the presence of an organophosphate, the biosensor comprising apolypeptide of the invention, and a means for detecting hydrolysis of anorganophosphate molecule by the polypeptide.

In yet another aspect, the present invention provides a method forscreening for agents which hydrolyse an organophosphate molecule, themethod comprising

-   -   (i) exposing the organophosphate to a candidate agent, and    -   (ii) measuring a fluorescent signal produced from step (i),        wherein the fluorescent signal is indicative of hydrolysis of        the organophosphate.

Preferably, the organophosphate is coumaphos or coroxon.

Further, it is preferred that the agent is a polypeptide or amicro-organism.

The polypeptide of the present invention can be mutated, and theresulting mutants screened for altered activity such as changes insubstrate specificity.

Thus, in a further aspect, the present invention provides a method ofproducing a polypeptide with enhanced ability to hydrolyse anorganophosphate or altered substrate specificity for an organophosphate,the method comprising

-   -   i) mutating one or more amino acids of a first polypeptide        according to the present invention,    -   ii) determining the ability of the mutant to hydrolyse an        organophosphate, and    -   iii) selecting a mutant with enhanced ability to hydrolyse the        organophosphate or altered substrate specificity for the        organophosphate, when compared to the first polypeptide.

As outlined in the Example section, this method has been successfullyapplied to produce the polypeptides provided as SEQ ID NO:2 and SEQ IDNO:3.

Preferably, the first polypeptide is selected from any one if SEQ IDNO's: 1 to 4.

In a further aspect, the present invention provides a polypeptideproduced according to the above method.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention will hereinafter be described by way of the followingnon-limiting Figures and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Structure of coumaphos and its hydrolysis products.

FIG. 2: The DNA sequence of opdA (SEQ ID NO:5). The region encoding thesignal peptide domain is given in bold, with the remaining sequencebeing referred to herein as SEQ ID NO:6.

FIG. 3: Amino acid sequence of OpdA (SEQ ID NO:1). The signal peptide isgiven in bold.

FIG. 4: Amino acid sequence alignment of OPD (SEQ ID NO:17) and OpdA.The secretion signals are given in bold.

KEY TO THE SEQUENCE LISTING

-   SEQ ID NO: 1—Polypeptide sequence of OpdA.-   SEQ ID NO: 2—Polypeptide sequence of OpdA minus the signal sequence.-   SEQ ID NO: 3—Polypeptide sequence of OpdA1.-   SEQ ID NO: 4—Polypeptide sequence of OpdA2.-   SEQ ID NO: 5—Polynucleotide sequence encoding OpdA.-   SEQ ID NO: 6—Polynucleotide sequence encoding OpdA minus the signal    sequence.-   SEQ ID NO: 7—Polynucleotide sequence encoding OpdA1.-   SEQ ID NO: 8—Polynucleotide sequence encoding OpdA2.-   SEQ ID NO's: 9 to 16—PCR primers.-   SEQ ID NO: 17—Polypeptide sequence of OPD from Flavobacterium sp.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless otherwise indicated, the recombinant DNA techniques utilized inthe present invention are standard procedures, well known to thoseskilled in the art. Such techniques are described and explainedthroughout the literature in sources such as, J. Perbal, A PracticalGuide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarbourLaboratory Press (1989), T. A. Brown (editor), Essential MolecularBiology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M.Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach,Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al.(Editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent).

Organophosphates

Organophosphates are synthetic organophosphorus esters and relatedcompounds such as phosphoroamidates. They have the general formula(RR′X)P═O or (RR′X)P═S, where R and R′ are short-chain groups. Forinsecticidal organophosphates X is a good leaving group, which is arequirement for the irreversible inhibition of acetylcholinesterase.

The polypeptides of the present invention hydrolyse the phosphoesterbonds of organophosphates. These organophosphates can be, but are notlimited to, oxon and thion OPs. The organophosphate can have aromatic oraliphatic leaving groups (X).

Although well known for their use as pesticides, organophosphates havealso been used as nerve gases against mammals. Accordingly, it isenvisaged that the polypeptides of the present invention will also beuseful for hydrolysis of organophosphates which are not pesticides.

Polypeptides

By “substantially purified polypeptide” we mean a polypeptide that hasgenerally been separated from the lipids, nucleic acids, otherpolypeptides, and other contaminating molecules with which it isassociated in its native state. Preferably, the substantially purifiedpolypeptide is at least 60% free, more preferably at least 75% free, andmost preferably at least 90% free from other components with which theyare naturally associated.

The % identity of a polypeptide is determined by FASTA (Pearson andLipman, 1988) analysis (GCG program) using the default settings and aquery sequence of at least 50 amino acids in length, and whereby theFASTA analysis aligns the two sequences over a region of at least 50amino acids. More preferably, the FASTA analysis aligns the twosequences over a region of at least 100 amino acids. More preferably,the FASTA analysis aligns the two sequences over a region of at least250 amino acids. Even more preferably, the FASTA analysis aligns the twosequences over a region of at least 350 amino acids.

Amino acid sequence mutants of the polypeptides of the present inventioncan be prepared by introducing appropriate nucleotide changes into anucleic acid sequence, or by in vitro synthesis of the desiredpolypeptide. Such mutants include, for example, deletions, insertions orsubstitutions of residues within the amino acid sequence. A combinationof deletion, insertion and substitution can be made to arrive at thefinal construct, provided that the final protein product possesses thedesired characteristics. Examples of mutants of the present inventionare provided in Example 8.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active site(s). Other sites of interestare those in which particular residues obtained from various species areidentical. These positions may be important for biological activity.These sites, especially those falling within a sequence of at leastthree other identically conserved sites, are preferably substituted in arelatively conservative manner. Such conservative substitutions areshown in Table 1 under the heading of “exemplary substitutions”.

Since the sequence of SEQ ID NO:1 is 90% identical to that of theFlavobacterium OPD enzyme it is possible that SEQ ID NO:1 could be usedto design mutants of the Flavobacterium OPD enzyme which have thedesired activity but are less than 90% identical. More specifically,those amino acids important for hydrolysing an organophosphate moleculecould be changed to match the polypeptides of the present invention andother amino acids not affecting this activity could also be changed toensure the identity levels do not exceed 90%. Examples of such OPDmutants include the amino acid changes L272F and/or H257Y. Such mutantsare also included in the present invention.

TABLE 1 Exemplary substitutions Original Exemplary Residue SubstitutionsAla (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; his; Asp (D) glu Cys(C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, ala His (H) asn; glnIle (I) leu; val; ala Leu (L) ile; val; met; ala; phe Lys (K) arg Met(M) leu; phe; Phe (F) leu; val; ala Pro (P) gly Ser (S) thr Thr (T) serTrp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe, ala

Furthermore, if desired, unnatural amino acids or chemical amino acidanalogues can be introduced as a substitution or addition into thepolypeptide of the present invention. Such amino acids include, but arenot limited to, the D-isomers of the common amino acids,2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid,2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogues in general.

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified during or aftersynthesis, e.g., by biotinylation, benzylation, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. These modifications may serve toincrease the stability and/or bioactivity of the polypeptide of theinvention.

Polypeptides of the present invention can be produced in a variety ofways, including production and recovery of natural proteins, productionand recovery of recombinant proteins, and chemical synthesis of theproteins. In one embodiment, an isolated polypeptide of the presentinvention is produced by culturing a cell capable of expressing thepolypeptide under conditions effective to produce the polypeptide, andrecovering the polypeptide. Effective culture conditions include, butare not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit protein production. An effective mediumrefers to any medium in which a cell is cultured to produce apolypeptide of the present invention. Such medium typically comprises anaqueous medium having assimilable carbon, nitrogen and phosphatesources, and appropriate salts, minerals, metals and other nutrients,such as vitamins. Cells of the present invention can be cultured inconventional fermentation bioreactors, shake flasks, test tubes,microtiter dishes, and petri plates. Culturing can be carried out at atemperature, pH and oxygen content appropriate for a recombinant cell.Such culturing conditions are within the expertise of one of ordinaryskill in the art.

Polynucleotides

By “isolated polynucleotide” we mean a polynucleotide which havegenerally been separated from the polynucleotide sequences with which itis associated or linked in its native state. Preferably, the isolatedpolynucleotide is at least 60% free, more preferably at least 75% free,and most preferably at least 90% free from other components with whichthey are naturally associated. Furthermore, the term “polynucleotide” isused interchangeably herein with the term “nucleic acid molecule”.

Polynucleotides of the present invention may possess one or moremutations when compared to SEQ ID NO's: 5 to 8. These mutations can bedeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagenesis on the nucleic acid). It is thus apparent thatpolynucleotides of the invention can be either naturally occurring orrecombinant.

The % identity of a polynucleotide is determined by FASTA (Pearson andLipman, 1988) analysis (GCG program) using the default settings and aquery sequence of at least 150 nucleotides in length, and whereby theFASTA analysis aligns the two sequences over a region of at least 150nucleotides. More preferably, the FASTA analysis aligns the twosequences over a region of at least 300 nucleotides. Even morepreferably, the FASTA analysis aligns the two sequences over a region ofat least 1050 nucleotides.

Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for the formation of a stable hybrid between anoligonucleotide and a complementary sequence on a nucleic acid moleculeof the present invention. The present invention includesoligonucleotides that can be used as, for example, probes to identifynucleic acid molecules or primers to produce nucleic acid molecules.

Oligonucleotides and/or polynucleotides of the present invention mayselectively hybridise to the sequences set out in SEQ ID NO's: 5 to 8under high stringency. As used herein, stringent conditions are thosethat (1) employ low ionic strength and high temperature for washing, forexample, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO₄ at 50° C.;(2) employ during hybridisation a denaturing agent such as formamide,for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin,0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer atpH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfateat 42° C. in 0.2×SSC and 0.1% SDS.

Vectors

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid.

One type of recombinant vector comprises a nucleic acid molecule of thepresent invention operatively linked to an expression vector. The phraseoperatively linked refers to the insertion of a nucleic acid moleculeinto an expression vector in a manner such that the molecule is able tobe expressed when transformed into a host cell. As used herein, anexpression vector is a DNA or RNA vector that is capable of transforminga host cell and effecting expression of a specified nucleic acidmolecule. Preferably, the expression vector is also capable ofreplicating within the host cell. Expression vectors can be eitherprokaryotic or eukaryotic, and are typically viruses or plasmids.Expression vectors of the present invention include any vectors thatfunction (i.e., direct gene expression) in recombinant cells of thepresent invention, including in bacterial, fungal, endoparasite,arthropod, other animal, and plant cells. Preferred expression vectorsof the present invention can direct gene expression in bacterial, yeast,plant and mammalian cells.

Expression vectors of the present invention contain regulatory sequencessuch as transcription control sequences, translation control sequences,origins of replication, and other regulatory sequences that arecompatible with the recombinant cell and that control the expression ofnucleic acid molecules of the present invention. In particular,recombinant molecules of the present invention include transcriptioncontrol sequences. Transcription control sequences are sequences whichcontrol the initiation, elongation, and termination of transcription.Particularly important transcription control sequences are those whichcontrol transcription initiation, such as promoter, enhancer, operatorand repressor sequences. Suitable transcription control sequencesinclude any transcription control sequence that can function in at leastone of the host cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin bacterial, yeast, plant and mammalian cells, such as, but not limitedto, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda,bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6,bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcoholoxidase, aiphavirus subgenomic promoters (such as Sindbis virussubgenomic promoters), antibiotic resistance gene, baculovirus,Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoonpoxvirus, other poxvirus, adenovirus, cytomegalovirus (such asintermediate early promoters), simian virus 40, retrovirus, actin,retroviral long terminal repeat, Rous sarcoma virus, heat shock,phosphate and nitrate transcription control sequences as well as othersequences capable of controlling gene expression in prokaryotic oreukaryotic cells. Additional suitable transcription control sequencesinclude tissue-specific promoters and enhancers.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed polypeptide of the present invention to be secretedfrom the cell that produces the polypeptide and/or (b) contain fusionsequences which lead to the expression of nucleic acid molecules of thepresent invention as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a protein of the present invention. Preferred signal segmentsinclude, but are not limited to, tissue plasminogen activator (t-PA),interferon, interleukin, growth hormone, histocompatibility and viralenvelope glycoprotein signal segments, as well as natural signalsequences. In addition, a nucleic acid molecule of the present inventioncan be joined to a fusion segment that directs the encoded protein tothe proteosome, such as a ubiquitin fusion segment. Recombinantmolecules may also include intervening and/or untranslated sequencessurrounding and/or within the nucleic acid sequences of nucleic acidmolecules of the present invention.

Host Cells

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained.

Suitable host cells to transform include any cell that can betransformed with a polynucleotide of the present invention. Host cellscan be either untransformed cells or cells that are already transformedwith at least one nucleic acid molecule (e.g., nucleic acid moleculesencoding one or more proteins of the present invention). Host cells ofthe present invention either can be endogenously (i.e., naturally)capable of producing proteins of the present invention or can be capableof producing such proteins after being transformed with at least onenucleic acid molecule of the present invention. Host cells of thepresent invention can be any cell capable of producing at least oneprotein of the present invention, and include bacterial, fungal(including yeast), parasite, arthropod, animal and plant cells.Preferred host cells include bacterial, mycobacterial, yeast, plant andmammalian cells. More preferred host cells include Agrobacterium,Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera,Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells(normal dog kidney cell line for canine herpesvirus cultivation), CRFKcells (normal cat kidney cell line for feline herpesvirus cultivation),CV-1 cells (African monkey kidney cell line used, for example, toculture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells.Particularly preferred host cells are E. coli, including E. coli K-12derivatives; Salmonella typhi; Salmonella typhimurium, includingattenuated strains; Spodoptera frugiperda; Trichoplusia ni; BHK cells;MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; andnon-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).Additional appropriate mammalian cell hosts include other kidney celllines, other fibroblast cell lines (e.g., human, murine or chickenembryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovarycells, mouse NIH/3T3 cells, LMTK cells and/or HeLa cells.

Recombinant DNA technologies can be used to improve expression oftransformed polynucleotide molecules by manipulating, for example, thenumber of copies of the polynucleotide molecules within a host cell, theefficiency with which those polynucleotide molecules are transcribed,the efficiency with which the resultant transcripts are translated, andthe efficiency of post-translational modifications. Recombinanttechniques useful for increasing the expression of polynucleotidemolecules of the present invention include, but are not limited to,operatively linking polynucleotide molecules to high-copy numberplasmids, integration of the polynucleotide molecules into one or morehost cell chromosomes, addition of vector stability sequences toplasmids, substitutions or modifications of transcription controlsignals (e.g., promoters, operators, enhancers), substitutions ormodifications of translational control signals (e.g., ribosome bindingsites, Shine-Dalgarno sequences), modification of polynucleotidemolecules of the present invention to correspond to the codon usage ofthe host cell, and the deletion of sequences that destabilizetranscripts. The activity of an expressed recombinant protein of thepresent invention may be improved by fragmenting, modifying, orderivatizing polynucleotide molecules encoding such a protein.

Transgenic Plants

As generally described in WO 99/53037, the levels of organophosphates ina sample can be reduced by exposing the sample to a transgenic plantexpressing a suitable enzyme. Typically, the sample is soil.Accordingly, the polynucleotide of the present invention can beexpressed in a transgenic plant, particularly the roots of the plant,for hydrolysing organophosphate molecules in the sample.

The term “plant” refers to whole plants, plant organs (e.g. leaves,stems roots, etc), seeds, plant cells and the like. Plants contemplatedfor use in the practice of the present invention include bothmonocotyledons and dicotyledons. Exemplary dicotyledons include cotton,corn, tomato, tobacco, potato, bean, soybean, and the like.

Transgenic plants, as defined in the context of the present inventioninclude plants (as well as parts and cells of said plants) and theirprogeny which have been genetically modified using recombinant DNAtechniques to cause or enhance production of at least one protein of thepresent invention in the desired plant or plant organ.

The polypeptide of the present invention may be expressed constitutivelyin the transgenic plants during all stages of development. Depending onthe use of the plant or plant organs, the proteins may be expressed in astage-specific manner. Furthermore, depending on the use, the proteinsmay be expressed tissue-specifically.

The choice of the plant species is determined by the intended use of theplant or parts thereof and the amenability of the plant species totransformation.

Regulatory sequences which are known or are found to cause expression ofa gene encoding a protein of interest in plants may be used in thepresent invention. The choice of the regulatory sequences used dependson the target plant and/or target organ of interest. Such regulatorysequences may be obtained from plants or plant viruses, or may bechemically synthesized. Such regulatory sequences are well known tothose skilled in the art.

Other regulatory sequences such as terminator sequences andpolyadenylation signals include any such sequence functioning as such inplants, the choice of which would be obvious to the skilled addressee.An example of such sequences is the 3′ flanking region of the nopalinesynthase (nos) gene of Agrobacterium tumefaciens.

Several techniques are available for the introduction of the expressionconstruct containing a DNA sequence encoding a protein of interest intothe target plants. Such techniques include but are not limited totransformation of protoplasts using the calcium/polyethylene glycolmethod, electroporation and microinjection or (coated) particlebombardment. In addition to these so-called direct DNA transformationmethods, transformation systems involving vectors are widely available,such as viral and bacterial vectors (e.g. from the genus Agrobacterium).After selection and/or screening, the protoplasts, cells or plant partsthat have been transformed can be regenerated into whole plants, usingmethods known in the art. The choice of the transformation and/orregeneration techniques is not critical for this invention.

Compositions

Compositions of the present invention include excipients, also referredto herein as “acceptable carriers”. An excipient can be any materialthat the animal, plant, plant or animal material, or environment(including soil and water samples) to be treated can tolerate. Examplesof such excipients include water, saline, Ringers solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal or o-cresol, formalin and benzylalcohol. Excipients can also be used to increase the half-life of acomposition, for example, but are not limited to, polymeric controlledrelease vehicles, biodegradable implants, liposomes, bacteria, viruses,other cells, oils, esters, and glycols.

Furthermore, the polypeptide of the present invention can be provided ina composition which enhances the rate and/or degree of organophosphatehydrolysis, or increases the stability of the polypeptide. For example,the polypeptide can be immobilized on a polyurethane matrix (Gordon etal., 1999), or encapsulated in appropriate liposomes (Petrikovics et al.2000a and b). The polypeptide can also be incorporated into acomposition comprising a foam such as those used routinely infire-fighting (LeJeune et al., 1998). As would be appreciated by theskilled addressee, the polypeptide of the present invention couldreadily be used in a sponge or foam as disclosed in WO 00/64539, thecontents of which are incorporated herein in their entirety.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal, plant, animal or plant material, orthe environment (including soil and water samples). As used herein, acontrolled release formulation comprises a composition of the presentinvention in a controlled release vehicle. Suitable controlled releasevehicles include, but are not limited to, biocompatible polymers, otherpolymeric matrices, capsules, microcapsules, microparticles, boluspreparations, osmotic pumps, diffusion devices, liposomes, lipospheres,and transdermal delivery systems. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into soil orwater which is in an area sprayed with an organophosphate pesticide. Theformulation is preferably released over a period of time ranging fromabout 1 to about 12 months. A preferred controlled release formulationof the present invention is capable of effecting a treatment preferablyfor at least about 1 month, more preferably for at least about 3 months,even more preferably for at least about 6 months, even more preferablyfor at least about 9 months, and even more preferably for at least about12 months.

The concentration of the polypeptide, vector, or host cell of thepresent invention that will be required to produce effectivecompositions for hydrolysing an organophosphate will depend on thenature of the sample to be decontaminated, the concentration of theorganophosphate in the sample, and the formulation of the composition.The effective concentration of the polypeptide, vector, or host cellwithin the composition can readily be determined experimentally, as willbe understood by the skilled artisan.

Biosensors

Biosensors are analytical devices typically consisting of a biologicallyactive material such as an enzyme and a transducer that converts abiochemical reaction into a quantifiable electronic signal that can beprocessed, transmitted, and measured. A general review of biosensorswhich have been used for the detection of orangophosphorus compounds isprovided by Rekha et al. (2000), the entire contents of which areincorporated by reference. The polypeptide of the present invention canbe adapted for use in such biosensors.

EXAMPLES Example 1 Enriching Soil Samples for Microorganisms withCoumaphos Hydrolytic Activity Fluorimetric Assay for CoumaphosHydrolysis

Phosphotriesterase enzymes catalyse the cleavage of a phosphoester bondin organophosphate (OP) molecules to yield a phosphodiester and analcohol: In the case of coumaphos (3-chloro-4-methyl-7-coumarinyldiethyl phosphorothioate), phosphotriesterase hydrolysis yieldsdiethylthiophosphate and the fluorescent alcohol, chlorferon(3-chloro-7-hydroxy-4-methyl coumarin; FIG. 1). Coumaphos hydrolysis cantherefore be measured fluorimetrically by the production of chlorferon,as measured by excitation at a wavelength of 355 nm and an emissionintensity of 460 nm. Chlorferon fluorescence was linear over the range0.01 μM to 2.5 μM.

All fluorescence measurements were performed in a POLARstar fluorimeter(BMG Technologies Pty Ltd, Australia) using 96-well white microtitreplates (FluoroNunc plates with PolySorp surface, Nalge NuncInternational) and final reaction volumes of 100 μl. Stock solutions ofcoumaphos and chlorferon (0.4 mM) were prepared in 20% methanol. Crudeassays of whole cells were performed in 100 μM coumaphos, 0.5% TritonX-100 and 50 mM Tris-HCl, pH8.0. Coumaphos hydrolytic assays of celllysates were performed without the Triton X-100.

The fluorescence of bacterial colonies and stained polyacrylamide gelswas examined using a hand-held long wavelength (approximately 340 nm) UVlight (Gelman Sciences).

Enrichment Cultures

The phosphodiester hydrolysis products of phosphotriesterases can beused as phosphorus sources by a wide range of bacteria (Cook et al.,1978; Rosenberg and Alexander, 1979). An enrichment culture wastherefore established in which 1 g of soil obtained from a domesticyard, which had previously been exposed to diazinon (a diethyl thionOP), served as an inoculum for 50 ml enrichment medium (Table 2), inwhich coumaphos was the only added phosphorus source.

TABLE 2 Composition of coumaphos enrichment media. Medium (per litre)Trace element solution (per litre) Tris 6.05 g (NH₄)₆Mo₇O₂₄•4H₂O 20 mgNH₄Cl 1 g H₃BO₃ 50 mg FeCl₂ 20 μg ZnCl₂ 30 mg KCl 0.5 g CoCl₂•6H₂O  3 mgSodium acetate 0.68 g MnCl₂•4H₂O 10 mg MgSO₄ 0.1 g Cupric acetate 10 mgp-aminobenzoate 0.9 mg nicotinic acid 0.9 mg Trace element solution 10ml Coumaphos 100 mM pH 7.0

Ten percent of the enrichment culture was subcultured twice into 50 mlfresh enrichment medium containing coumaphos as the sole phosphorussource. Coumaphos was then replaced with diazinon (at 100 mM) in theenrichment medium and the culture subcultured as before. After 3 daysincubation at 28° C., the enrichment culture was further subculturedinto media in which 100 mM parathion (another diethyl thion OP) was thesole phosphorus source. It was noted that after two days the culture hadturned yellow (presumably due to the production of p-nitrophenol). Thisculture was then diluted in phosphorus-free medium and plated onto lowsalt LB plates (10 g/l tryptone, 5 g/l yeast extract and 2.5 g/l NaCl).After three days of growth at 28° C., approximately 100 colonies werepicked randomly, re-streaked to ensure purity and then assayed forcoumaphos hydrolytic activity using the microtitre plate assay describedabove. Fluorescence was measured after 8 hours at room temperature. Oneisolate (designated P230) demonstrated significant fluorescence and thisisolate was examined further. Colonies of this isolate also demonstratedfluorescence on an agar plate containing coumaphos.

Example 2 Identification of Isolate P230

Isolate P230 was a Gram negative, catalase positive and oxidasepositive, rod-shaped bacterium. To determine the identity of isolateP230, sequence analysis of the 16S rRNA gene was performed. DNA wasextracted from isolate P230 according to the method of Rainey et al.(1992). Cells of a P230 culture that had been grown in low salt LBmedium (2 ml) overnight at 28° C. were pelleted by centrifugation in amicrofuge (12 000 rpm/2 minutes). The cell pellet was resuspended in 400μl STE buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH8.0) and 5 μlof a freshly-prepared lysozyme solution (0.3 μg/μl) was added. Afterincubation at 37° C. for 20 minutes, Proteinase K (15 μl of a 1%solution) and SDS (10 μl of a 25% solution) were added and the reactionsincubated at 60° C. for 30 minutes. DNA preparations were then extractedsequentially with an equal volume of buffer-saturated phenol, and thenan equal volume of chloroform.

The 16S rRNA gene was amplified from the extracted DNA by PCR usingbacterial universal primers 27f (5′ AGAGTTTGATCMTGGCTCAG 3′) (SEQ ID NO:9) and 1492r (5′ TACGGYTACCTTGTTACGACTT 3′) (SEQ ID NO: 10), the namesof which are based on the numbering system of the E. coli 16S rRNA gene(Lane, 1991). Approximately 1320 by of the 16S rRNA gene from isolateP230 was obtained and sequence similarities were performed using theFASTA algorithm (Pearson and Lipman, 1988). The 16S rRNA gene of isolateP230 was very similar in sequence to that of other Agrobacterium strains(Table 3).

TABLE 3 Nucleic acid sequence comparisons of the 16S rRNA genes ofisolate P230 with those of various Agrobacterium strains. Agrobacteriumstrains Sequence Identity (%) Agrobacterium radiobacter LMG383 100Agrobacterium sp LMG11936 99.7 Agrobacterium sp. MSMC211 99.5Agrobacterium sp. LMG11915 99.3

These results suggested that isolate P230 was an Agrobacterium strain.The utilization of carbon sources by isolate P230 was examined using theBiolog system (Oxoid), according to procedures recommended by themanufacturer. The carbon utilization profile was then compared with thatof known species of Agrobacterium (Table 4; Krieg and Holt, 1984). Theisolate was capable of using sucrose, ornithine and glucose as carbonsources. This, along with a positive oxidase reaction determined usingKovac's method (Kovac, 1956), suggested that the isolate was mostsimilar to either A. tumefaciens biovar1 or A. radiobacter biovar1.

TABLE 4 Carbon utilization profiles and oxidase status of isolate P230and known Agrobacterium species. Agrobacterium spp. Carbon A. A. A. A.A. source/ tumefaciens tumefaciens radiobacter radiobacter rhizogenesIsolate test biovar1 biovar2 biovar1 biovar2 biovar2 A. rubi P230 Tween80 − − − − − − − Sucrose + − + − − − + Ornithine + + + + + + +D-glucose + + + + + + + Oxidase + − + − − − + test (Kovac)

A. tumefaciens biovar1 and A. radiobacter biovar1 can be distinguishedby the presence of a tumour-inducing plasmid in the former. Thetumour-inducing ability of strain P230 was tested in a tomato seedlingby transferring a heavy suspension of bacteria in water to the leavesand using a sterile needle to pierce the surface of the leaves throughthe suspension. No evidence of tumours was seen after a period of fourweeks. A. tumefaciens C58 was used as a positive control and producedtumours in this period of time. A cured strain of A. tumefaciens C58 wasused as a negative control and produced the same effects in the testplant as isolate P230. Therefore isolate P230 was designated as a strainof Agrobacterium radiobacter biovar1.

Example 3 Constitutive Expression of Coumaphos Hydrolytic Activity

In order to determine if the phosphotriesterase activity of A.radiobacter P230 was constitutively expressed, regardless of thepresence of OPs, the parathion hydrolytic activities of cultures of A.radiobacter P230 were examined in the presence and absence of parathion(Table 5). Growth was monitored by measuring the optical density of thecultures at 595 nm in a BioRad Model 3550-UV microplatespectrophotometer. Parathion hydrolytic activity was assayed accordingto the procedure of Serdar et al. (1989). This involved measuring theformation of p-nitrophenol from parathion at 405 nm in a BioRad Model3550-UV microplate spectrophotometer. The reaction mixture contained 880μM parathion in 50 mM Tris-HCl pH8.0 (this reaction also contained 5%methanol). Table 5 shows that parathion hydrolytic activity wasconstitutively expressed in isolate P230 and that the majority of thisactivity was expressed in early- to mid-log phase.

Example 4 Native Polyacrylamide Gel Electrophoresis (PAGE) of P230Extracts

To demonstrate that a single enzyme was involved in coumaphoshydrolysis, native gels of A. radiobacter P230 cell extracts werestained for coumaphos hydrolytic activity. A culture (50 ml) of A.radiobacter P230 in low salt LB broth was pelleted by centrifugation at8000 g for 15 minutes and the cell pellet resuspended in 2 ml 50 mMTris-HCl pH8.0. Cells were disrupted by sonication (five 15 secondbursts at 4° C.) and large cell debris or intact cells removed bycentrifugation (8000 g for 15 minutes). An aliquot of the resultantsupernatant (containing 5 μg protein) was then separated on a 10% (29:1acrylamide:bis) SDS-PAGE gel. Prior to loading, neither SDS norβ-mercaptoethanol was added to the sample and furthermore, the samplewas not boiled as in conventional SDS-PAGE. After electrophoresis thegel was equilibrated for 5 min in 50 mM Tris-HCl pH8.0, and thenincubated for a further 5 min in 50 mM Tris-HCl pH8.0, containing 8 μMcoumaphos. The gel was then examined under UV light as described above.A major fluorescent band was detected, indicating that the P230 isolatecontains a single enzyme with coumaphos hydrolytic activity. This enzymehad an apparent molecular mass of 66 kDa.

TABLE 5 Parathion hydrolytic activities of P230 cultures grown in thepresence or absence of parathion to an OD at 595 nm of 0.280. Parathionhydrolytic activity (μmol/min/mg Culture protein) +parathion 3.36 ± 0.18−parathion 3.13 ± 0.07

Example 5 Cloning the Gene Responsible for Coumaphos Hydrolytic ActivityCloning Techniques and DNA Preparations

General cloning techniques, unless otherwise indicated, were standardand as described by Sambrook et al. (1989). Chromosomal DNA wasextracted from A. radiobacter P230 according to the method of Gardineret al. (1996). Briefly, an overnight culture (100 ml) of A. radiobacterP230 was pelleted by centrifugation at 5000 g for 20 minutes, washedtwice with 10 ml ice-cold STE buffer (see above), and finallyresuspended in 10 ml STE. Lysozyme (20 mg) was added and the cellsincubated for 2 hours at 37° C. An equal volume of STE was added alongwith SDS (to a final concentration of 2% [w/v]) and RNase (to a finalconcentration of 20 μg/ml) and the cell lysate incubated at 42° C. for 1hour. Proteinase K (50 μg/ml final concentration) was then added and thelysate incubated at 55° C. until the solution became translucent. Anequal volume of buffer-saturated phenol/chloroform (1:1) was added, thesample thoroughly mixed and then centrifuged at 5000 g for 1 hour at 4°C. The upper aqueous layer was transferred to a clean tube using abroken pipette in order to prevent shearing of DNA. To precipitate thechromosomal DNA, 3M sodium acetate, pH5.2, (0.1 volume) and ice-coldethanol (2.5 volumes) were added, the solution mixed gently and placedat −20° C. for 1 hour. The DNA was then removed using a “hooked” pasteurpipette. This precipitate was washed with 70% ethanol and air-dried for5 minutes. TE buffer (pH8.0; 1 ml) was added and the DNA left todissolve at 4° C. overnight.

Library Construction in E. coli

A partial Sau3Al digest of A. radiobacter P230 chromosomal DNA wasprepared by digesting 37.5 μg of DNA with 4, 2, 1, 0.5 and 0.25 units ofSau3Al restriction endonuclease for 10 minutes at 37° C. DNA fragmentsin the size range of 10-12 kb were excised from a 0.7% agarose gel andextracted using a QIAGEN PCR purification/gel extraction kit, accordingto the manufacturer's instructions. pBluescript KS+ plasmid DNA(Stratagene), prepared using the Geneworks Ultraclean Plasmid miniprepkit, was digested with BamHI for 1 hour at 37° C. and dephosphorylatedusing calf intestinal alkaline phosphatase (Boehringer Mannheim). Thephosphatase was then removed using the QIAquick PCR purification kit(QIAGEN). The size-fractionated P230 DNA fragments were ligated to theBamHI digested, phosphatase treated pBluescript vector, using T4 DNAligase and the T4 ligase buffer provided by New England Biolabs.Ligations were performed for 20 hours at 4° C.

The ligated DNA was then transformed into E. coli DH10β using therevised Hanahan transformation method (Sambrook et al., 1989). Thetransformation mix was plated onto LB agar plates containing ampicillin(100 μg/ml), X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside; 40μg/ml) and IPTG (isopropyl-β-D-thiogalactoside; 40 μg/ml), and incubatedat 37° C. overnight. Approximately 350 white colonies were revealed.

Triparental Matings for Expression in A. tumefaciens

Since it was possible that the gene encoding the OP hydrolytic activityin A. radiobacter would not be expressed sufficiently in E. coli toallow identification on the basis of expression, the pBluescriptplasmids from the E. coli library above were transferred into aclosely-related strain, A. tumefaciens C58. A. tumefaciens C58 is anon-OP hydrolysing strain of Agrobacterium (Zimmerer at al., 1966).Briefly, the white colonies identified above on LB plates containingampicillin, X-Gal and IPTG were patched onto LB plates, without anyadditions. Also patched onto the same plates at the same places were A.tumefaciens C58 and E. coli JM109 (Yanisch-Perron et al., 1985)containing the conjugative, cointegrative plasmid, pR751::Tn813 (Bowenand Pemberton, 1985). It was intended that the conjugative cointegrativeplasmid would move into E. coli DH10β containing thepBluescript-derivatives and form a cointegrate. The cointegrates wouldthen be transferred into A. tumefaciens C58.

The tri-parental matings were incubated overnight at 28° C. The matingmixtures were then scraped up and assayed for coumaphos hydrolyticactivity in a microtitre plate. This involved resuspending the matingmix in an assay buffer containing 0.5% Triton X-100, 100 μM coumaphosand 50 mM Tris-HCl pH8.0. The microtitre plates were then incubated for8 hours at room temperature, and the amount of fluorescence noted asdescribed above. One mating mix (containing clone p65) demonstratedsignificant fluorescence.

Coumaphos Hydrolytic Activity of E. coli DH10β p65 Cell-Free Extracts

Cell-free extracts of E. coli DH10β p65, and control extracts containingthe pBluescript vector alone, were prepared from cells grown to mid-logphase on LB medium containing ampicillin (100 μg/ml). The 50 ml cultureswere pelleted by centrifugation at 8000 g for 15 minutes and resuspendedin 2 ml 50 mM Tris-HCl pH8.0. The cells were disrupted by sonication(five 15 second bursts at 4° C.) and large cell debris or intact cellswere removed by centrifugation (8000 g for 15 minutes). Aliquots(containing 15 μg protein) of the supernatants were assayed forcoumaphos hydrolytic activity. The increase in fluorescence over timewas measured and the amount of activity determined. It can be seen fromTable 6 that cell-free extracts of E. coli DH10β containing clone p65displayed significant coumaphos hydrolytic activity compared to that ofthe vector-only controls.

Localisation of the Gene Encoding the OP Hydrolytic Activity in Clonep65

Clone p65 DNA was digested to completion with HindIII and the resultantfour fragments [5.5 kb (containing pBluescript vector), 4 kb, 3.5 kb and1.4 kb] separated and subsequently excised from a 1% agarose gel. Thefragments were extracted using the QIAquick PCR purification kit(QIAGEN) and ligated to HindIII digested pBluescript DNA prepared asdescribed above. The ligation mixes were transformed into E. coli DH10βand individual clones assayed for coumaphos hydrolytic activity. Severalof the clones containing the 4 kb HindIII fragment demonstratedcoumaphos hydrolytic activity, depending on the orientation of thefragment in pBluescript relative to the lac promoter.

TABLE 6 Coumaphos hydrolytic activity of cell-free extracts of E. coliDH10β p65 and control extracts containing the pBluescript vector alone.Coumaphos hydrolytic activity (nmol/min/mg Strain protein) E. coli DH10β(pBluescript) 0.78 ± 0.04 E. coli DH10β (p65) 3.30 ± 0.07

Example 6 Sequence of opdA

The nucleotide sequence of the 4 kb HindIII fragment identified abovewas determined using primers complementary to the T3 and T7 promoters inthe vector and ‘primer walking’. DNA was sequenced using the BigDyeTerminator system (Applied BioSystems) on the Applied BioSystems ABIPRISM 377 automated DNA sequencer. An open reading frame (ORF) wasidentified in the same orientation as the lacZ promoter in clonespossessing activity and in the opposite orientation in clones lackingactivity. The open reading frame contains 1152 nucleotides (FIG. 2) and,when translated, would encode a protein of 384 amino acids (FIG. 3) and41.4 kDa.

Sequence similarities were calculated using the FASTA algorithm (Pearsonand Lipman, 1988). This indicated that the ORF had 88% nucleotidesequence identity to opd, a previously identified phosphotriesterasegene from Flavobacterium sp. ATCC27551 (Mulbry and Karns, 1989).Furthermore, the inferred amino acid sequence of the ORF was 90%identical to that of the Flavobacterium OPD enzyme (FIG. 4). For thisreason we have named this open reading frame ‘opdA’.

Some notable differences were observed between the Flavobacterium opdsequence and that of opdA from A. radiobacter P230 (FIGS. 2 and 3).There appears to be one less amino acid in the putative signal sequenceof the OpdA protein and the signal cleavage site is also different.Furthermore, a frameshift near the 3′ end of the opdA gene gives OpdA anadditional 16 amino acids. This region has been sequenced multiple timesto ensure that the extra base in opdA is not a sequencing error.

The native OPD enzyme is a homodimer that contains two zinc ions permonomeric subunit (Benning et al., 1995). The two His residues atpositions 254 and 257 in the OPD protein sequence are located near thebimetallic active site present in each monomer and are thought tointeract with active site residues and the substrate in the substratebinding pocket. Replacement of each of these His residues with Arg andLeu, respectively, resulted in enzymes possessing only two metal atomsper dimer (diSioudi et al., 1999). The OpdA protein has Arg and Tyr atthe positions corresponding to His 254 and His257 in OPD (FIG. 4). Itwould therefore be expected that the OpdA native enzyme would containonly two metal ions per dimer rather than four, as in native OPD.

Example 7 Activity of the Purified OpdA Protein

To confirm that the open reading frame in FIG. 3 encoded the proteinresponsible for OP hydrolytic activity, the protein was expressed andpurified as a fusion protein with maltose-binding protein.

Expression of OpdA and OPD as Fusion Proteins

The OpdA and OPD proteins were expressed in Escherichia coli using thepMAL protein fusion and purification system of New England Biolabs,which results in the expression of maltose-binding protein (MBP) fusionproteins.

To clone the opdA gene into the pMAL-c vector, the opdA gene (withoutthe signal peptide domain) was amplified by the polymerase chainreaction (PCR) using the upstream and downstream primers,5′GATCGTCTGCAGCCAATCGGTACAGGCGATCTG (SEQ ID NO: 11) and5′GATCGTAAGCTTTCATCGTTCGGTATCTTGACGGGGAAT (SEQ ID NO: 12), respectively.A PstI cloning site was inserted at the start codon and a HindIIIcloning site at the stop codon (underlined bases). The PCR fragment wassubsequently cloned into the PstI-HindIII cloning sites of pMAL-c, togenerate the recombinant plasmid, pmal-opdA.

The opd gene (Mulbry and Karns, 1989) was cloned into the pMAL-c2×vector (New England Biolabs) in a similar way. The opd gene, without thesignal peptide domain, was amplified using PCR. The upstream anddownstream oligonucleotide primers, 5′GATCGTGGATCCTCGATCGGCACAGGCGATCGG(SEQ ID NO: 13) and 5′GATCGTAAGCTTTCATGACGCCCGCAAGGTCGG (SEQ ID NO: 14),respectively, were designed to contain a BamHI restriction site at theopd start codon and a HindIII restriction site at the stop codon(underlined bases). The PCR fragment was subsequently cloned into theBamHI-HindIII restriction sites of pMAL-c2× to generate the recombinantplasmid, pFmal.

Purification of OpdA and OPD Proteins

Both MBP fusion proteins were expressed in E. coil DH10β cells. Optimalproduction of MBP fusion proteins was obtained when mid-log cells(OD₆₀₀=0.6) were induced with 0.1 mM isopropyl-β-D-thiogalactopyranosidefor 5 hours at 37° C. Harvested cells were disrupted by sonication andthe soluble fraction loaded onto an amylose resin (New England Biolabs),equilibrated with 50 mM Tris-HCl pH7.5. MBP fusion proteins were elutedwith 10 mM maltose in 50 mM Tris-HCl pH7.5. Fractions containingcoumaphos hydrolytic activity were pooled and cleaved with Xa protease(10 μg/ml; New England Biolabs) for 5 hours. The cleaved fractions werethen passaged through a DEAE sepharose ion exchange resin. Cleaved OpdAand OPD proteins did not bind to this resin and eluted with the voidvolume. Fractions from this sample appeared to be pure as judged bySDS-PAGE. The amount of protein in purified samples was calculatedaccording to the method of Gill and von Hippel (1989).

Kinetic Analyses of OPD and OpdA (i) Substrates

Kinetic parameters were determined for the hydrolysis by OpdA and OPD ofthe following substrates: coumaphos, parathion (O,O-diethylp-nitrophenyl phosphorothioate; Riedel de Haan), parathion-methyl(O,O-dimethyl p-nitrophenyl phosphorothioate; Riedel de Haan), paraoxon(O,O-diethyl p-nitrophenyl phosphate; Sigma), coroxon(3-chloro-4-methyl-7-coumarinyl diethyl phosphate; Alltech), fenthion(O,O-dimethyl O-[3-methyl 4-(methylthio)phenyl]phosphorothioate; Riedelde Haan), diazinon(labelled—O,O-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidinyl)-phosphorothioate;Alltech), dMUP (O,O-dimethyl 4-methyl-umbelliferyl phosphate; a giftfrom Alan Devonshire), chlorpyrifos (O,O-diethylO-3,5,6-trichloro-2-pyridyl phosphorothioate; Alltech) and phosmet(S-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl O,O-dimethylphosphorodithioate; Alltech).

(ii) Assays

All reactions contained organophosphates dissolved in methanol, exceptfor phosmet, which was dissolved in acetone. The concentration ofacetone or methanol in the reactions was constant at 5%, whereappropriate. All reactions were performed in 50 mM Tris-HCl pH 8.0 at25° C.

The initial rates of reactions of purified OPD and OpdA with coumaphosand coroxon were determined using the fluorimetric assay describedabove.

The initial rates of reaction of both OPD and OpdA with dMUP weredetermined using a fluorimetric assay to quantitate the formation of thehydrolysis product, 4-methyl umbelliferone (Roth, 1969). Thefluorescence was measured using an excitation wavelength of 355 nm andan emission intensity of 460 nm.

The initial rates of reaction of both purified enzymes with parathion,parathion-methyl and paraoxon were measured spectrophotometrically byquantitating the formation of the hydrolysis product, p-nitrophenol, at405 nm and using an extinction coefficient of 17 000 M⁻¹cm⁻¹ (Dumas etal., 1989b).

The initial rates of reaction of OpdA with fenthion were quantitatedspectrophotometrically by monitoring a reduction in absorbance at 252 nm(Ibrahim and Cavagnol, 1966). A lack of hydrolysis of fenthion andphosmet by OPD was confirmed by thin layer chromatography after 24 hourincubation of the substrate with OPD.

The reaction rates of OpdA and OPD with chlorpyrifos were measuredspectrophotometrically by monitoring the increase in absorbance at 276nm (Dumas et al., 1989b).

The hydrolysis of phosmet was measured by quantitating the formation offree thiols during the course of the reaction using DTNB (Ellman'sreagent; 5′5 dithio-bis-(2-nitro benzoic acid)) as has been describedpreviously for monitoring P—S hydrolysis in organophosphates (Lai etal., 1995). This involved the addition of DTNB (80 μl of 1 mg/ml in 50mM sodium phosphate pH7.5 and methanol, 1:1 (v/v)) to 20 μl aliquots ofthe reaction taken at various times.

The hydrolysis of diazinon was monitored using radiolabelled diazinon(ethyl-1-¹⁴C; 14.8 MBq/mmol) in the radiometric partition assaypreviously used for radiolabelled OP substrates (Campbell et al., 1998).At various times during the reaction, an aliquot (50 μl) was removed anddiluted with 150 μl water. This was then extracted with 500 μldichloromethane. The upper aqueous phase (150 μl) was removed andquantitated by liquid scintillation.

Results

Results of the kinetic analyses are given in Table 7. OpdA and OPD wereable to hydrolyse the substrates coumaphos, coroxon, paraoxon,parathion, parathion-methyl, diazinon, chlorpyrifos and dMUP. OpdA had ahigher k_(cat) for both parathion-methyl and dMUP, and OPD was unable tohydrdolyse either phosmet or fenthion. A lack of hydrolysis of thelatter two substrates was also observed over a 24 hour period by thinlayer chromatography (Munnecke and Hsieh, 1976). This involved theextraction of a 100 μl reaction containing 0.4 mM substrate with anequal volume of ethyl acetate. The upper organic phase was gently driedwith a nitrogen stream, and the remaining residue was dissolved in 10 μlacetone and then applied to a neutral silica gel F₂₅₄ TLC plate(Alltech, NSW, Australia). The plate was then developed inhexane-chloroform-methanol (7:2:1) and compounds visualised by shortwavelength ultra-violet light. Hydrolysis of both phosmet and fenthionwere consistently observed for OpdA and no hydrolysis was seen for OPD.

In summary, several differences in substrate specificity between OpdAand OPD were observed. OpdA hydrolysed fenthion and phosmet whereas OPDdid not. Furthermore, there was a significant difference between OpdAand OPD in the k_(cat) values for dimethyl OPs, with OpdA possessing ahigher k_(cat) for methyl-parathion and dMUP than OPD. We would alsoexpect OpdA, like OPD (Dumas et al., 1989a; Yang et al, 1995), tohydrolyse OP nerve agents.

As discussed above, the two His residues at positions 254 and 257 in theOPD protein sequence are located near the bimetallic active site presentin each monomer and are thought to interact with active site residuesand the substrate in the substrate binding pocket (Benning et al.,1995). Replacement of each of these His residues with Arg and Leu,respectively, resulted in enzymes with only one metal ion per monomer,increased catalytic activity for larger substrates such as demeton, anddecreased activity for smaller

TABLE 7 Kinetic parameters of purified OpdA and OPD enzymes for variousOP substrates. K_(m) (μM) k_(cat) (min⁻¹) Substrate/Structure OpdA OPDOpdA OPD

8.3 ± 1.8 21.4 ± 6.0  12.4 ± 0.6  14.1 ± 2.6 

15.9 ± 1.9  25.3 ± 1.3  22.7 ± 0.1  39.5 ± 5.3 

242 ± 61  225 ± 14  33.5 ± 0.5  46.0 ± 0.4 

92.6 ± 6.4  50.6 ± 12.2 21.9 ± 2.0  23.5 ± 0.2 

61.2 ± 2.3  32.9 ± 1.7  94.2 ± 0.8  5.46 ± 0.05

208.3 ± 13.2  — 0.100 ± 0.002 —

148.6 ± 17.2  — 1.63 ± 0.01 — . . . /cont.

51.9 ± 4.5  54.2 ± 5.4  65.2 ± 6.7  56.5 ± 2.9 

32.6 ± 8.1  47.2 ± 3.0  0.525 ± 0.005 0.90 ± 0.01

66.0 ± 9.1  46.7 ± 2.8  81.7 ± 9.1  20.5 ± 2.3 substates like paraoxon (diSioudi et al., 1999). It was postulated thatchanges in the number of bound metal ions may enhance structuralflexibility and improve access of larger substrates to the active site,while simultaneously decreasing activity for smaller substrates. TheOpdA protein has Arg and Tyr at the positions corresponding to His 254and His257 in OPD (FIG. 4). It is therefore surprising that OpdApossessed a higher k_(cat) for methyl-parathion than OPD, yet itsk_(cat) for the larger ethyl-parathion substrate was similar to that ofOPD. Similar results were obtained for the coumaphos/dMUP substratepair. Clearly, differences between the OpdA and OPD amino acid sequencesother than those at residues 253/254 and 256/257 affect catalyticactivity.

Example 8 Identification of OpdA Mutants with Altered Specificity

The plasmid pmal-opdA was transformed into the E. coli mutator strainXL1-red. The plasmid was propagated in this strain for 120 generations,with plasmid extractions occurring after every 24 generations. Theseplasmids were then transformed into E. coli DH1013 and thetransformation mix diluted to 50 ml in LB containing ampicillin.

When the culture reached an OD₅₉₅ of 0.3, fusion protein expression wasinduced with 0.1 mM IPTG, and induction allowed to occur for 5 hours.The culture was then pelleted by centrifugation, resuspended in 2 ml ofsterile 50 mM Tris-HCl pH7.5 with the addition of malathion to a finalconcentration of 440 μM. This assay mixture was left for 1 hour and thehydrolysis of malathion detected using Ellman's reagent (DTNB) (Lai etal., 1995).

Pools containing activity were then diluted and plated onto LB plateswith ampicillin. Individual colonies were then selected and tested formalathion and dimethoate hydrolytic activity as described above. Twocolonies (designated pmal-opdA1 and pmal-opdA2) were selected andexamined further.

The sequences of the two mutants were examined and compared with that ofwild-type OpdA. OpdA1 contained 4 mutations (P42S, P134S, A170S andS237G) (SEQ ID NO: 3) and OpdA2 contained one mutation (A119D) (SEQ IDNO: 4). The numbering system is based on OpdA numbering of amino acidresidues, taking into account the signal sequence. To correlate thenumbering with OPD, add one to each number.

OpdA and the two mutants OpdA1 and OpdA2 were purified after expressionin the plasmid pCY76. The genes were amplified by PCR using the primerspETopdA5 (5′GATCGTGAATTC CATATGCCAATCGGTACA, with EcoRI site underlinedand NdeI double underlined) (SEQ ID NO: 15) and pETopdA3(5′GATCGTGGATCCTCATCGTTCGGTATCTTG, with BamHI site underlined) (SEQ IDNO:16). PCR fragments were digested with EcoRI and BamHI and ligatedwith similarly-digested pBluescript. Sequence of the fragments wereconfirmed in this vector. The pBS-derivatives were then digested withNdel-BamHI and ligated with Ndel-Bg/II-digested pCY76. Positive cloneswere grown in 500 ml of LB. After the cultures had grown for 24 hours,they were pelleted by centrifugation at 7000 g, 15 minutes at 4° C. Thepellets were resuspended in 4 ml 50 mM Tis-HCl pH7.5 and broken bysonication (Harcourt et al., 2002). Cell-free extracts were then chargedonto a DEAE sepharose column that was pre-equilibrated with 50 mMTris-HCl pH7.5. OpdA and variants did not bind to this column and theeluant was collected and placed on a heparin sepharose columnpre-equilibrated with 50 mM Tris-HCl pH7.5 (Pharmacia). OpdA andvariants were bound by this column and eluted with 50 mM Tris-HClpH7.5/0.1 M NaCl. After this column step, OpdA was judged to be pure bySDS-PAGE. The kinetics of the proteins were examined against thealiphatic OPs, dimethoate, malathion, malaoxon and DFP (diisopropylfluorophosphate) (Table 8). Both mutants were active against dimethoate,malathion and malaoxon, whereas the wild-type OpdA was not. Furthermore,the mutants had increased activity for DFP compared to that of wild-typeOpdA.

TABLE 8 The kinetic parameters of purified OpdA and the mutants, OpdA1and OpdA2, for various OP substrates. K_(m) (μM) k_(cat) (min⁻¹)Substrate OpdA OpdA1 OpdA2 OpdA OpdA1 OpdA2

2.3 ± 0.4 18.1 ± 0.6  9.6 ± 5.6 1.36 ± 0.04 45.9 ± 0.9  27.5 ± 0.7 

nd¹ 78.9 ± 17.7 14.3 ± 4.2  nd 1.22 ± 0.07 1.4 ± 0.1

nd 33.3 ± 14.2 40.9 ± 7.2  nd 1.21 ± 0.07 1.43 ± 0.07

nd 159.2 ± 28.2  45.7 ± 8.9  nd 1.98 ± 0.05 1.84 ± 0.06 ¹nd = notdetected

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed above are incorporated herein in theirentirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed in Australia before thepriority date of each claim of this application.

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1-37. (canceled)
 38. A host cell comprising a recombinant polynucleotidewhich encodes a polypeptide comprising amino acids whose sequence is atleast 95% identical to the sequence set forth as SEQ ID NO:2.
 39. Thehost cell of claim 38, wherein the polypeptide comprises amino acidswhose sequence is at least 97% identical to the sequence set forth asSEQ ID NO:2.
 40. The host cell of claim 38, wherein the polypeptide is asubstitution mutant of the polypeptide comprising amino acids whosesequence is set forth as SEQ ID NO:2.
 41. The host cell of claim 38,wherein the polynucleotide comprises nucleotides whose sequence is atleast 95% identical to the sequence set forth as SEQ ID NO:6.
 42. Thehost cell of claim 38, wherein the polynucleotide is comprised in avector.
 43. The host cell of claim 42, wherein the vector is a plasmidvector.
 44. The host cell of claim 42, wherein the vector is integratedinto one or more host cell chromosomes.
 45. The host cell of claim 38which is a bacterial cell or a yeast cell, or an extract thereofcomprising the polypeptide.
 46. The host cell of claim 45, wherein thebacterial cell is Salmonella sp., Escherichia sp., Bacillus sp., orListeria sp., or an extract thereof comprising the polypeptide.
 47. Thehost cell of claim 45, wherein the bacterial cell is Escherichia coli,or an extract thereof comprising the polypeptide.
 48. The host cell ofclaim 39 which is a bacterial cell or a yeast cell, or an extractthereof comprising the polypeptide.
 49. The host cell of claim 48,wherein the bacterial cell is Salmonella sp., Escherichia sp., Bacillussp., or Listeria sp., or an extract thereof comprising the polypeptide.50. The host cell of claim 48, wherein the bacterial cell is Escherichiacoli, or an extract thereof comprising the polypeptide.
 51. The hostcell of claim 40 which is a bacterial cell or a yeast cell, or anextract thereof comprising the polypeptide.
 52. The host cell of claim51, wherein the bacterial cell is Salmonella sp., Escherichia sp.,Bacillus sp., or Listeria sp., or an extract thereof comprising thepolypeptide.
 53. The host cell of claim 51, wherein the bacterial cellis Escherichia coli, or an extract thereof comprising the polypeptide.54. A composition comprising the host cell of claim 38 and one or moreacceptable carriers.
 55. A composition comprising the host cell of claim51 and one or more acceptable carriers.
 56. A composition comprising thehost cell of claim 52 and one or more acceptable carriers.
 57. Acomposition comprising the host cell of claim 53 and one or moreacceptable carriers.
 58. A process for preparing a polypeptidecomprising amino acids whose sequence is at least 95% identical to thesequence set forth as SEQ ID NO:2, the process comprising cultivatingthe host cell of claim 38 under conditions which allow production of thepolypeptide, and extracting the polypeptide.